Nucleic acid delivery

ABSTRACT

The present invention provides formulations and methods to enhance the delivery of nucleic acids to cells. Formulations comprising dextrin polymers in combination with sugars provide enhanced delivery of nucleic acids, particularly eucaryotic expression vectors, demonstrate enhanced delivery of nucleic acids to cells in vivo. The present invention also provides methods of treatment in combination with such formulations.

RELATION TO OTHER APPLICATIONS

The present application claims the benefit of U.S. Patent ProvisionalApplication Ser. No. 60/245,539 filed Nov. 3, 2000 and U.S. ProvisionalApplication Ser. No.60/287,871 filed Apr. 30, 2001 pursuant to 35 U.S.C.§119(e).

FIELD OF THE INVENTION

The invention relates to a method for the delivery of nucleic acid tocells, particularly but not exclusively, in gene therapy, includingcompositions the osmolarity of which corresponds substantially to thephysiological osmolarity which surrounds the cell or tissue

BACKGROUND OF THE INVENTION

Many methods have been developed over the last 30 years to facilitatethe introduction of nucleic acid into cells which have greatly assisted,inter alia, our understanding of the control of gene expression.

Conventional methods to introduce DNA into cells are well known in theart and typically involve the use of chemical reagents, cationic lipidsor physical methods. Chemical methods which facilitate the uptake of DNAby cells include the use of DEAE-Dextran (Vaheri and Pagano, Science175:434). DEAE-dextran associates with and introduces the DNA intocells. However this can result in loss of cell viability. Calciumphosphate is also a commonly used chemical agent which, whenco-precipitated with DNA, introduces the DNA into cells (Graham et alVirology (1973) 52: 456).

The use of cationic lipids (e.g. liposomes (Felgner (1987) Proc. Natl.Acad. Sci USA, 84:7413) has become a common method since it does nothave the degree of toxicity shown by the above described chemicalmethods. The cationic head of the lipid associates with the negativelycharged nucleic acid backbone of the DNA to be introduced. The lipid/DNAcomplex associates with the cell membrane and fuses with the cell tointroduce the associated DNA into the cell. Liposome mediated DNAtransfer has several advantages over existing methods. For example,cells which are recalcitrant to traditional chemical methods are moreeasily transfected using liposome mediated transfer.

More recently still, physical methods to introduce DNA have becomeeffective means to reproducibly transfect cells. Direct microinjectionis one such method which can deliver DNA directly to the nucleus of acell (Capecchi (1980) Cell, 22:p 479). This allows the analysis ofsingle cell transfectants. So called “biolistic” methods physicallyinsert DNA into cells and/or organelles using a high velocity particlescoated with DNA (Neumann (1982) EMBO J, 1: 841).

Electroporation is arguably the most popular method to transfect DNA.The method involves the use of a high voltage electrical charge tomomentarily permeabilise cell membranes making them permeable tomacromolecular complexes. However physical methods to introduce DNA doresult in considerable loss of cell viability due to intracellulardamage. These methods therefore require extensive optimisation and alsorequire expensive equipment.

More recently still a method termed immunoporation has become arecognised techinque for the introduction of nucleic acid into cells,see Bildirici et al Nature (2000) 405, 298. The technique involves theuse of beads coated with an antibody to a specific receptor. Thetransfection mixture includes nucleic acid, typically vector DNA,antibody coated beads and cells expressing a specific cell surfacereceptor. The coated beads bind the cell surface receptor and when ashear force is applied to the cells the beads are stripped from the cellsurface. During bead removal a transient hole is created through whichnucleic acid and/or other biological molecules can enter. Transfectionefficiency of between 40-50% is achievable depending on the nucleic acidused.

Typically, gene therapy involves the transfer, and optionally the stableinsertion, of new genetic information into cells for the therapeutictreatment of disease. Genes that have been successfully expressed inmice after transfer by retrovirus vectors include human hypoxanthinephosphoribosyl transferase (Miller A et al, 1984, Science 255:630).Bacterial genes have also been transferred into mammalian cells, in theform of bacterial drug resistance genes. Transformation of hematopoieticprogenitor cells to drug resistance by eukaryotic virus vectors has alsoaccomplished with recombinant retrovirus based vector systems (Hock R Aand Miller A D 1986, Nature 320:275-277; Joyner, et al. (1983) Nature305:556-558; Williams D A et al 1984, Nature 310:476-480; Dick J E etal, 1985, Cell 42:71-79); Keller G et al 1985, Nature 318: 149-154;Eglitis M et al, 1985, Science 230: 1395-1398). Adeno-associated virusvectors have been used successfully to transduce mammalian cell lines toneomycin resistance (Hermonat P L and Muzyczka N, 1984, supra; TratschinJ D et al, 1985, Mol. Cell. Biol. 5:3251). Other viral vector systemsthat have been investigated for use in gene transfer includepapovaviruses and vaccinia viruses (See Cline, M L (1985) Pharmac. Ther.29:69-92).

The main issues with respect to gene therapy relate to the efficienttargeting of nucleic acid to cells and the establishment of high leveltransgene expression in selected tissues. A number of methodologies havebeen developed which purport to facilitate either or both of theserequirements. For example, U.S. Pat. No. 6,043,339 discloses the use ofsignal peptides which when fused to nucleic acid, can facilitate thetranslocation of the linked nucleic acid across cell membranes. U.S.Pat. No. 6,083,714 discloses a combined nucleic acid and targettingmeans which uses the polycation poly-lysine coupled to an integrinreceptor thereby targetting cells expressing the integrin. EP1013770discloses the use of nuclear localisation signals (NLS) coupled tooligonucleotides. The conjugate may be covalently linked to vector DNAand the complex used to transfect cells. The NLS sequence serves tofacilitate the passage of the vector DNA across the nuclear membranethereby targetting gene delivery to the nucleus.

Nucleic acid, for example vector DNA, may be introduced into an animalvia a variety of routes including enterally (orally, rectally orsublingually) or parenterally (intravenously, subcutaneously, or byinhalation).

It is known that introduction of certain aqueous solutions into theperitoneal cavity can be useful in the treatment of patients sufferingfrom renal failure. Such treatment is known as peritoneal dialysis. Thesolutions contain electrolytes similar to those present in plasma; theyalso contain an osmotic agent, normally dextrose, which is present in aconcentration sufficient to create a desired degree of osmotic pressureacross the peritoneal membrane. Under the influence of this osmoticpressure, an exchange takes place across the peritoneal membrane andresults in withdrawal from the bloodstream of waste products, such asurea and creatinine, which have accumulated in the blood due to the lackof normal kidney function. While this exchange is taking place, there isalso a net transfer of dextrose from the solution to the blood acrossthe peritoneal membrane, which causes the osmolality of the solution tofall. Because of this, the initial osmolality of the solution must bemade fairly high (by using a sufficiently high concentration ofdextrose) in order that the solution continues to effect dialysis for areasonable length of time before it has to be withdrawn and replaced byfresh solution.

Other osmotic agents have been proposed for use in peritoneal dialysisand in recent years dextrin (a starch hydrolysate polymer of glucose)has been used. When instilled in the peritoneal cavity, dextrin isslowly absorbed via the lymphatic system, eventually reaching theperipheral circulation. The structure of dextrin is such that amylasesbreak the molecule down into oligosaccharides in the circulation. Theseare cleared by further metabolism into glucose.

Typically, a medium chosen to introduce gene therapy materials to apatient via a body cavity might be a buffered saline solution, forinstance, phosphate buffered saline (PBS).

Dextrin solutions have been proposed as the medium for delivery of drugsto the body via the peritoneum. In GB-A-2207050, such a solution isproposed for the intraperitoneal administration of drugs for whichenteral administration is unsatisfactory. Such an approach is stated tobe particularly useful for the delivery of peptide drugs such aserythropoetin and growth hormones. Reference is also made tocephalosporin antibiotics. Dextrin solutions have also been describedfor the administration of chemotherapeutic agents in the treatment ofovarian cancers. The use of icodextrin formulations to increase theefficacy of chemotherapeutics (especially 5FU) by increasing their dwelltime in the peritoneal space is well described in Dobbie J W. (1997) AdvPerit Dial. 13:162-7 and McArdle C S, et al. (1994) Br J Cancer70(4):762-6.

The present invention is directed to a dextrin containing solution whichshows enhanced ability to deliver nucleic acid to cells resulting inhigh level expression of transfected genes.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical formulation comprising anucleic acid in a solution comprising dextrin and at least one sugar,the osmolarity of said solution corresponding substantially tophysiological osmolarity.

The present invention further provides a method to deliver a nucleicacid to a cell wherein the nucleic acid is carried in a solutioncomprising dextrin and at least one sugar, the osmolarity of whichcorresponds substantially to the physiological osmolarity of the milieusurrounding the cell.

The invention further provides a method of treating an mammal with anucleic acid wherein the nucleic acid is administered to said mammal ina pharmaceutical formulation comprising dextrin and at least one sugar.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a histogram providing measuring the expression of thebeta-galactosidase gene in epithelial tissues of the peritoneum inrabbits following the intraperitoneal instillation of a 100 ul of asolution containing a recombinant adenoviral vector encodingbeta-galactosidase in various formulations as more fully described inExample 1 herein. The vertical axis represents the levels of viral RNAin tissues as determined by RT-PCR.

FIG. 2 is a graphical representation of the results of the murinexenograft prostate cancer model described in Example 2 herein. Thevertical axis represents cumulative survival as a fraction of thestarting number of animals remaining alive. The horizontal graphrepresents time in Days.

FIG. 3 is a graphical representation of the data generated from themurine model of ovarian cancer more fully described in Example 4 herein.drawings. Formulation H/Treatment Group 2 is represented by X,Formulation I/Treatment Group 1 is represented by squares, FormulationJ/Treatment Group 3 is represented by circles; and FormulationL/Treatment Group 4 is represented by triangles.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a pharmaceutical formulation comprising anucleic acid in a solution comprising dextrin and at least one sugar,the osmolarity of said solution corresponding substantially tophysiological osmolarity.

Dextrin:

The term “dextrin” means a glucose polymer which is produced by thehydrolysis of starch and which consists of glucose units linked togetherby means mainly of α-1,4 linkages. Typically dextrins are produced bythe hydrolysis of starch obtained from various natural products such aswheat, rice, maize and tapioca. In addition to α-1,4 linkages there maybe a proportion of α-1,6 linkages in a particular dextrin, the amountdepending on the starch starting material. Since the rate ofbiodegradability of α-1,6 linkages is typically less than that for α-1,4linkages, for many applications it is preferred that the percentage ofα-1,6 linkages is less than 10% and preferably less than about 5%.

Any dextrin is a mixture of polyglucose molecules of different chainlengths. As a result, no single number can adequately characterise themolecular weight of such a polymer. Accordingly various averages areused,.the most common being the weight average molecular weight (Mw) andthe number average molecular weight (Mn). Mw is particularly sensitiveto changes in the high molecular weights content of the polymer whilstMn is largely influenced by changes in the low molecular weight of thepolymer. It the preferred practice of the invention, the Mw of thedextrin is in the range from about 1,000 to 200,000, more preferablyfrom about 2,000 to 55,000.

The term “degree of polymerisation” (DP) can also be used in connectionwith polymer mixtures. For a single polymer molecule, DP means thenumber of polymer units. For a mixture of molecules of different DP's,weight average DP and number average DP correspond to Mw and Mn. Inaddition DP can also be used to characterise a polymer by referring tothe polymer mixture having a certain percentage of polymers of DPgreater than a particular number or less than a particular number. It ispreferred that, in the present invention, the dextrin contains more thanabout 15% of polymers of DP greater than 12 and, more preferably, morethan about 50% of polymers of DP greater than 12.

Preferably the dextrin is present in the solution in an amount of lessthan about 20%. Preferably the dextrin is present in the solution in anamount selected from about: 1% (w/v); 2% (w/v); 3% (w/v); 4% (w/v); 5%(w/v); 6% (w/v); 7% (w/v); 8% (w/v); 9% (w/v); 10% (w/v); 11w/v; 12%w/v; 13% w/v; 14% w/v; 15% w/v; 16% w/v; 17% w/v; 18% w/v; 19% w/v; 20%w/v. More preferably the dextrin is present from about 2 to 5% byweight, most preferably about 4% by weight.

Physiological Osmolarity:

As indicated, the solution possesses an osmolarity essentially isotonicwith the osmolarity of the physiological milieu of the cell to betreated. Generally, the physiological osmolarity maintained in mammalianbody cavities is approximately 330 milliosmolar and will vary somewhatwith the particular body cavity. For example, an isotonic solution forinstillation in the human peritoneal cavity would have an osmolarity ofapproximately 290-300 milliosmolar. In the preferred practice of theinvention for intraperitoneal instillation, the solution will possess anosmolarity from about 250 to 350 milliosmolar, more preferably fromabout 275 to 330 milliosmolar. The adjustments to maintain approximateisotonic osmolarity depending on the particular physiological milieuwill be readily apparent to those of skill in the art.

Sugar:

The term “sugar” refers to a monosaccharide, disaccharide oroligosaccharide. Monosaccharides have the empirical formula (CH₂O)_(n)wherein n=3 or greater. Examples of monosaccharides, which are merelymeant to be illustrative and not restrictive, are glucose, galactose,mannose, allose, altrose, gulose, idose, talose, fructose. Disaccharidesconsist of two monosaccharides linked by a glycoside bond.Non-restrictive examples of dissacharides are sucrose, maltose,cellobiose, gentiobiose, lactose. Typically oligosaccharides are sugarswith more than two monosaccharide units. Non-restrictive examples ofoligosaccharides are raffinose and melezitose. Sugars can be naturallyoccuring or industrially manufactured.

In a preferred method of the invention the sugar is a disaccharide. Morepreferably still the amount of disaccharide is between about 1-10% w/v.Preferably the amount of disaccharide is between about 2-5% w/v. In apreferred embodiment of the invention, the disaccharide is sucrose in isabout 3% w/v.

In a further method of the invention the amount of dextrin is aboutbetween 2%-20% w/v and the amount of sucrose is about 1-10% w/v.

More ideally still the amount of dextrin is about 4% w/v and the amountof sucrose is about 3% w/v.

It will be apparent that the exact combination will depend on theosmolarity of the fluid surrounding the cell or tissue. Typically, it isdesirable to maintain an isosmotic equilibrium between the solutionincluding the nucleic acid and the fluid surrounding the cells/tissues.

Divalent Cation:

More preferably still said solution further comprises a divalent cation.Preferably the divalent cation is at least 0.2 mM. More preferably stillthe divalent cation concentration is between about 0.2-3.0 mM. Ideallythe divalent cation is MgCl₂ and the concentration is about 2.0 mM.Alternatively the divalent cation is provided by CaCl₂.

Preferably the solution comprises about 4% w/v dextrin, about 3% w/vsucrose and about 2.0 mM MgCl₂. Alternatively, the dextrin concentrationis about 15% w/v.

It will be apparent to one skilled in the art that the solution hasutility with respect to the in vitro delivery of nucleic acid to cellsfor the recombinant production of polypeptides encoded by the nucleicacid. The invention also encompasses gene therapy, both the in vivointroduction of nucleic acid into cells and ex vivo introduction ofnucleic acid into cells followed by introduction of transfected cellsinto an animal in need of gene therapy.

Nucleic Acid:

Preferably said nucleic acid molecule is adapted for eukaryoticexpression. Typically said adaptation includes, by example and not byway of limitation, the provision of transcription control sequences(promoter sequences) which mediate cell/tissue specific expression.These promoter sequences may be cell/tissue specific, inducible orconstitutive.

“Promoter” is an art recognised term and, for the sake of clarity,includes the following features which are provided by example only, andnot by way of limitation. Enhancer elements are cis acting nucleic acidsequences often found 5′ to the transcription initiation site of a gene(enhancers can also be found 3′ to a gene sequence or even located inintronic sequences). Enhancers function to increase the rate oftranscription of the gene to which the enhancer is linked. Enhanceractivity is responsive to trans acting transcription factors(polypeptides) which have been shown to bind specifically to enhancerelements. The binding/activity of transcription factors (please seeEukaryotic Transcription Factors, by David S Latchman, Academic PressLtd, San Diego) is responsive to a number of physiological/environmentalcues which include, by example and not by way of limitation,intermediary metabolites (eg glucose, lipids), environmental effectors(eg light, heat,).

Promoter elements also include so called TATA box and RNA polymeraseinitiation selection (RIS) sequences which function to select a site oftranscription initiation. These sequences also bind polypeptides whichfunction, inter alia, to facilitate transcription initiation selectionby RNA polymerase.

Adaptations also include the provision of selectable markers andautonomous replication sequences which facilitate the maintenance ofsaid vector in either the eukaryotic cell or prokaryotic host. Vectorswhich are maintained autonomously are referred to as episomal vectors.Episomal vectors are desirable since these molecules can incorporatelarge DNA fragments (30-50 kb DNA). Episomal vectors of this type aredescribed in WO98/07876.

Adaptations which facilitate the expression of vector encoded genesinclude the provision of transcription termination/polyadenylationsequences. This also includes the provision of internal ribosome entrysites (IRES) which function to maximise expression of vector encodedgenes arranged in bi-cistronic or multi-cistronic expression cassettes.Expression control sequences also include so-called Locus ControlRegions (LCRs). These are regulatory elements which conferposition-independent, copy number-dependent expression to linked geneswhen assayed as transgenic constructs. LCRs include regulatory elementsthat insulate transgenes from the silencing effects of adjacentheterochromatin, Grosveld et al., Cell (1987), 51: 975-985.

Expression control sequences also encompass, ubiquitous chromatinopening elements (UCOE's), see WO/GB00/05393. UCOE's are nucleic acidelements that are responsible for establishing an open chromatinstructure across a locus that consists exclusively of ubiquitouslyexpressed, housekeeping genes. These elements are not derived from anLCR. A UCOE is a polynucleotide which opens chromatin or maintainschromatin in an open state and facilitates reproducible expression of anoperably-linked gene in cells of at least two different tissue types.

These adaptations are well known in the art. There is a significantamount of published literature with respect to expression vectorconstruction and recombinant DNA techniques in general. Please see,Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory, Cold Spring Harbour, N.Y. and referencestherein; Marston, F (1987) DNA Cloning Techniques: A Practical ApproachVol III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

Vectors are typically viral based and may be derived from virusesincluding adenovirus; retrovirus; adeno-associated virus; herpesvirus;lentivirus; vaccinia virus; and baculovirus.

The terms “therapeutic virus” and “therapeutic viral vector” are usedinterchangeably herein to refer to viruses used as therapeutic agents(e.g. wild-type viruses, attenuated viruses), vaccine vectors orrecombinant viruses containing modifications to the genome to enhancetherapeutic effects. The use of viruses or “viral vectors” astherapeutic agents are well known in the art as previously discussed.Additionally, a number of viruses are commonly used as vectors for thedelivery of exogenous genes. Commonly employed vectors includerecombinantly modified enveloped or non-enveloped DNA and RNA viruses,preferably selected from baculoviridiae, parvoviridiae, picornoviridiae,herpesveridiae, poxviridae, adenoviridiae, or picornnaviridiae. Chimericvectors may also be employed which exploit advantageous elements of eachof the parent vector properties (See e.g., Feng, et al. (1997) NatureBiotechnology 15:866-870). Such viral vectors may be wild-type or may bemodified by recombinant DNA techniques to be replication deficient,conditionally replicating or replication competent.

Preferred vectors are derived from the adenoviral, adeno-associatedviral and retroviral genomes. In the most preferred practice of theinvention, the vectors are derived from the human adenovirus genome.Particularly preferred vectors are derived from the human adenovirusserotypes 2 or 5. The replicative capacity of such vectors may beattenuated (to the point of being considered “replication deficient”) bymodifications or deletions in the E1a and/or E1b coding regions. Othermodifications to the viral genome to achieve particular expressioncharacteristics or permit repeat administration or lower immune responseare preferred. Most preferred are human adenoviral type 5 vectorscontaining a DNA sequence encoding the p53 tumor suppressor gene. In themost preferred practice of the invention as exemplified herein, thevector is replication deficient vector adenoviral vector encoding thep53 tumor suppressor gene A/C/N/53 as described in Gregory, et al., U.S.Pat. No. 5,932,210 issued Aug. 3, 1999 (the entire teaching of which isherein incorporated by reference).

Alternatively, the viral vectors may be conditionally replicating orreplication competent. Conditionally replicating viral vectors are usedto achieve selective expression in particular cell types while avoidinguntoward broad spectrum infection. Examples of conditionally replicatingvectors are described in Pennisi, E. (1996) Science 274:342-343;Russell, and S. J. (1994) Eur. J. of Cancer 30A(8):1165-1171. Additionalexamples of selectively replicating vectors include those vectorswherein an gene essential for replication of the virus is under controlof a promoter which is active only in a particular cell type or cellstate such that in the absence of expression of such gene, the viruswill not replicate. Examples of such vectors are described in Henderson,et al., U.S. Pat. No. 5,698,443 issued Dec. 16, 1997 and Henderson, etal., U.S. Pat. No. 5,871,726 issued Feb. 16, 1999 the entire teachingsof which are herein incorporated by reference.

Additionally, the viral genome may be modified to include induciblepromoters which achieve replication or expression only under certainconditions. Examples of inducible promoters are known in the scientificliterature (See, e.g. Yoshida and Hamada (1997) Biochem. Biophys. Res.Comm. 230:426-430; Iida, et al. (1996) J. Virol. 70(9):6054-6059; Hwang,et al.(1997) J. Virol 71(9):7128-7131; Lee, et al. (1997) Mol. Cell.Biol. 17(9):5097-5105; and Dreher, et al.(1997) J. Biol. Chem 272(46);29364-29371.

The viruses may also be designed to be selectively replicating viruses.Particularly preferred selectively replicating viruses are described inRamachandra, et al. PCT International Publication No. WO 00/22137,International Application No. PCT/US99/21452 published Apr. 20, 2000 andHowe, J., PCT International Publication No. WO WO0022136, InternationalApplication No. PCT/US99/21451 published Apr. 20, 2000. A particularlypreferred selectively replicating recombinant adenovirus is the virusdl01/07/309 as more fully described in Howe, J.

It has been demonstrated that viruses which are attenuated forreplication are also useful in the therapeutic arena For example theadenovirus dl1520 containing a specific deletion in the E1b55K gene(Barker and Berk (1987) Virology 156: 107) has been used withtherapeutic effect in human beings. Such vectors are also described inMcCormick (U.S. Pat. No. 5,677,178 issued Oct. 14, 1997) and McCormick,U.S. Pat. No. 5,846,945 issued Dec. 8, 1998. The method of the presentinvention may also be used in combination with the administration ofsuch vectors to minimize the pre-existing or induced humoral immuneresponse to such vectors.

Additionally, the therapeutic virus may incorporate a therapeutictransgene for expression in an infected cell. The term “therapeutictransgene” refers to a nucleotide sequence the expression of which inthe target cell produces a therapeutic effect. The term therapeutictransgene includes but is not limited to tumor suppressor genes,antigenic genes, cytotoxic genes, cytostatic genes, pro-drug activatinggenes, apoptotic genes, pharmaceutical genes or anti-angiogenic genes.The vectors of the present invention may be used to produce one or moretherapeutic transgenes, either in tandem through the use of IRESelements or through independently regulated promoters.

The term “tumor suppressor gene” refers to a nucleotide sequence, theexpression of which in the target cell is capable of suppressing theneoplastic phenotype and/or inducing apoptosis. Examples of tumorsuppressor genes useful in the practice of the present invention includethe p53 gene, the APC gene, the DPC-4 gene, the BRCA-1 gene, the BRCA-2gene, the WT-1 gene, the retinoblastoma gene (Lee, et al. (1987) Nature329:642), the MMAC-1 gene, the adenomatous polyposis coli protein(Albertsen, et al., U.S. Pat. No. 5,783,666 issued Jul. 21, 1998), thedeleted in colon carcinoma (DCC) gene, the MMSC-2 gene, the NF-1 gene,nasopharyngeal carcinoma tumor suppressor gene that maps at chromosome3p21.3. (Cheng, et al. 1998. Proc. Nat. Acad. Sci. 95:3042-3047), theMTS1 gene, the CDK4 gene, the NF-1 gene, the NF2 gene, and the VHL gene.A particularly preferred adenovirus for therapeutic use is the A/C/N/53vector encoding the p53 tumor suppressor gene as more fully described inGregory, et al., U.S. Pat. No. 5,932,210 issued Aug. 3, 1999, the entireteaching of which is herein incorporated by reference.

The term “antigenic genes” refers to a nucleotide sequence, theexpression of which in the target cells results in the production of acell surface antigenic protein capable of recognition by the immunesystem. Examples of antigenic genes include carcinoembryonic antigen(CEA), p53 (as described in Levine, A. PCT International Publication No.WO94/02167 published Feb. 3, 1994). In order to facilitate immunerecognition, the antigenic gene may be fused to the MHC class I antigen.Preferably the antigenic gene is derived from a tumour cell specificantigen. Ideally a tumour rejection antigen. Tumour rejection antigensare well known in the art and include, by example and not by way oflimitation, the MAGE, BAGE, GAGE and DAGE families of tumour rejectionantigens, see Schulz et al Proc Natl Acad Sci USA, 1991, 88, pp 991-993.

It has been known for many years that tumour cells produce a number oftumour cell specific antigens, some of which are presented at the tumourcell surface. These are generally referred to as tumour rejectionantigens and are derived from larger polypeptides referred to as tumourrejection antigen precursors. Tumour rejection antigens are presentedvia HLA's to the immune system. The immune system recognises thesemolecules as foreign and naturally selects and destroys cells expressingthese antigens. If a transformed cell escapes detection and becomesestablished a tumour develops. Vaccines have been developed based ondominant tumour rejection antigen's to provide individuals with apreformed defence to the establishment of a tumour.

The term “cytotoxic gene” refers to nucleotide sequence, the expressionof which in a cell produces a toxic effect. Examples of such cytotoxicgenes include nucleotide sequences encoding pseudomonas exotoxin, ricintoxin, diptheria toxin, and the like.

The term “cytostatic gene” refers to nucleotide sequence, the expressionof which in a cell produces an arrest in the cell cycle. Examples ofsuch cytostatic genes include p21, the retinoblastoma gene, the E2F-Rbgene, genes encoding cyclin dependent kinase inhibitors such as P16,p15, p18 and p19, the growth arrest specific homeobox (GAX) gene asdescribed in Branellec, et al. (PCT Publication WO97/16459 published May9, 1997 and PCT Publication WO96/30385 published Oct. 3, 1996).

The term “cytokine gene” refers to a nucleotide sequence, the expressionof which in a cell produces a cytokine. Examples of such cytokinesinclude GM-CSF, the interleukins, especially IL-1, IL-2, IL-4, IL-12,IL-10, IL-19, IL-20, interferons of the α, β and γ subtypes, consensusinterferons and especially interferon α-2b and fusions such asinterferon α-2α-1.

The term “chemokine gene” refers to a nucleotide sequence, theexpression of which in a cell produces a cytokine. The term chemokinerefers to a group of structurally related low-molecular cytokines weightfactors secreted by cells are structurally related having mitogenic,chemotactic or inflammatory activities. They are primarily cationicproteins of 70 to 100 amino acid residues that share four conservedcysteine. These proteins can be sorted into two groups based on thespacing of the two amino-terminal cysteines. In the first group, the twocysteines are separated by a single residue (C-x-C), while in the secondgroup, they are adjacent (C—C). Examples of member of the ‘C-x-C’chemokines include but are not limited to platelet factor 4 (PF4),platelet basic protein (PBP), interleukin-8 (IL-8), melanoma growthstimulatory activity protein (MGSA), macrophage inflammatory protein 2(MIP-2), mouse Mig (m119), chicken 9E3 (or pCEF-4), pig alveolarmacrophage chemotactic factors I and II (AMCF-I and -II), pre-B cellgrowth stimulating factor (PBSF), and IP10. Examples of members of the‘C—C’ group include but are not limited to monocyte chemotactic protein1 (MCP-1), monocyte chemotactic protein 2 (MCP-2), monocyte chemotacticprotein 3 (MCP-3), monocyte chemotactic protein 4 (MCP-4), macrophageinflammatory protein 1α (MIP-1-α), macrophage inflammatory protein 1β(MIP-1-β), macrophage inflammatory protein 1-γ (MIP-1-γ), macrophageinflammatory protein 3α (MIP-3-α, macrophage inflammatory protein 3β(MIP-3-β), chemokine (ELC), macrophage inflammatory protein-4 (MIP-4),macrophage inflammatory protein 5 (MIP-5), LD78β, RANTES, SIS-epsilon(p500), thymus and activation-regulated chemokine (TARC), eotaxin,I-309, human protein HCC-1/NCC-2, human protein HCC-3, mouse proteinC10.

The term “pharmaceutical protein gene” refers to nucleotide sequence,the expression of which results in the production of protein havepharmaceutically effect in the target cell. Examples of suchpharmaceutical genes include the proinsulin gene and analogs (asdescribed in PCT International Patent Application No. WO98/31397, growthhormone gene, dopamine, serotonin, epidermal growth factor, GABA, ACTH,NGF, VEGF (to increase blood perfusion to target tissue, induceangiogenesis, PCT publication WO98/32859 published Jul. 30, 1998),thrombospondin etc. Also, the pharmaceutical protein gene may encompassimmunoreactive proteins such as antibodies, Fab fragments, Fv fragments,humanized antibodies, chimeric antibodies, single chain antibodies, andhuman antibodies derived from non-human sources.

The term “pro-apoptotic gene” refers to a nucleotide sequence, theexpression thereof results in the induction of the programmed cell deathpathway of the cell. Examples of pro-apoptotic genes include p53,adenovirus E3-11.6 K(10.5 K), the adenovirus E4orf4 gene, p53 pathwaygenes, and genes encoding the caspases.

The term “pro-drug activating genes” refers to nucleotide sequences, theexpression of which, results in the production of protein capable ofconverting a non-therapeutic compound into a therapeutic compound, whichrenders the cell susceptible to killing by external factors or causes atoxic condition in the cell. An example of a prodrug activating gene isthe cytosine deaminase gene. Cytosine deaminase converts5-fluorocytosine to 5 fluorouracil, a potent antitumor agent). The lysisof the tumor cell provides a localized burst of cytosine deaminasecapable of converting 5FC to 5FU at the localized point of the tumorresulting in the killing of many surrounding tumor cells. This resultsin the killing of a large number of tumor cells without the necessity ofinfecting these cells with an adenovirus (the so-called bystandereffect”). Additionally, the thymidine kinase (TK) gene (see e.g. Woo, etal. U.S. Pat. No. 5,631,236 issued May 20, 1997 and Freeman, et al. U.S.Pat. No. 5,601,818 issued Feb. 11, 1997) in which the cells expressingthe TK gene product are susceptible to selective killing by theadministration of gancyclovir may be employed.

The term “anti-angiogenic” genes refers to a nucleotide sequence, theexpression of which results in the extracellular secretion ofanti-angiogenic factors. Anti-angiogenesis factors include angiostatin,inhibitors of vascular endothelial growth factor (VEGF) such as Tie 2(as described in PNAS(USA)(1998) 95:8795-8800), endostatin.

It will be readily apparent to those of skill in the art thatmodifications and or deletions to the above referenced genes so as toencode functional subfragments of the wild type protein may be readilyadapted for use in the practice of the present invention. For example,the reference to the p53 gene includes not only the wild type proteinbut also modified p53 proteins. Examples of such modified p53 proteinsinclude modifications to p53 to increase nuclear retention, deletionssuch as the Δ13-19 amino acids to eliminate the calpain consensuscleavage site (Kubbutat and Vousden (1997) Mol. Cell. Biol. 17:460-468,modifications to the oligomerization domains (as described in Bracco, etal. PCT published application WO97/0492 or U.S. Pat. No. 5,573,925,etc.).

It will be readily apparent to those of skill in the art that the abovetherapeutic genes may be secreted into the media or localized toparticular intracellular locations by inclusion of a targeting moietysuch as a signal peptide or nuclear localization signal (NLS). Alsoincluded in the definition of therapeutic transgene are fusion proteinsof the therapeutic transgene with the herpes simplex virus type 1(HSV-1) structural protein, VP22. Fusion proteins containing the VP22signal, when synthesized in an infected cell, are exported out of theinfected cell and efficiently enter surrounding non-infected cells to adiameter of approximately 16 cells wide. This system is particularlyuseful in conjunction with transcriptionally active proteins (e.g. p53)as the fusion proteins are efficiently transported to the nuclei of thesurrounding cells. See, e.g.Elliott, G. & O'Hare, P. Cell.88:223-233:1997; Marshall, A. & Castellino, A. Research News Briefs.Nature Biotechnology. 15-205:1997; O'Hare, et al. PCT publicationWO97/05265 published Feb. 13, 1997. A similar targeting moiety derivedfrom the HIV Tat protein is also described in Vives, et al. (1997) J.Biol. Chem. 272:16010-16017.

It may be valuable in some instances to utilize or design vectors toachieve introduction of the exogenous transgene in a particular celltype. Certain vectors exhibit a natural tropism for certain tissuetypes. For example, vectors derived from the genus herpesviridiae havebeen shown to have preferential infection of neuronal cells. Examples ofrecombinantly modified herpesviridiae vectors are disclosed in U.S. Pat.No. 5,328,688 issued Jul. 12, 1994. Cell type specificity or cell typetargeting may also be achieved in vectors derived from viruses havingcharacteristically broad infectivities by the modification of the viralenvelope proteins. For example, cell targeting has been achieved withadenovirus vectors by selective modification of the viral genome knoband fiber coding sequences to achieve expression of modified knob andfiber domains having specific interaction with unique cell surfacereceptors. Examples of such modifications are described in Wickham, etal.(1997) J. Virol 71(11):8221-8229 (incorporation of RGD peptides intoadenoviral fiber proteins); Arnberg, et al. (1997) Virology 227:239-244(modification of adenoviral fiber genes to achieve tropism to the eyeand genital tract); Harris and Lemoine (1996) TIG 12(10):400-405;Stevenson, et al.(1997) J. Virol. 71(6):4782-4790; Michael, et al.(1995)Gene Therapy 2:660-668 (incorporation of gastrin releasing peptidefragment into adenovirus fiber protein); and Ohno, et al.(1997) NatureBiotechnology 15:763-767 (incorporation of Protein A-IgG binding domaininto Sindbis virus). Other methods of cell specific targeting have beenachieved by the conjugation of antibodies or antibody fragments to theenvelope proteins (see, e.g. Michael, et al. (1993) J. Biol. Chem268:6866-6869, Watkins, et al. (1997) Gene Therapy 4:1004-1012; Douglas,et al. (1996) Nature Biotechnology 14: 1574-1578. Alternatively,particularly moieties may be conjugated to the viral surface to achievetargeting (See, e.g. Nilson, et al. (1996) Gene Therapy 3:280-286(conjugation of EGF to retroviral proteins)). Additionally, the virallyencoded therapeutic transgene also be under control of a tissue specificpromoter region allowing expression of the transgene preferentially inparticular cell types.

Vectors may also be non-viral and are available from a number ofcommercial sources readily available to the man-skilled in the art. Forexample the vectors may be plasmids which can be episomal or integratingplasmids.

In a further preferred embodiment of the invention said nucleic acid isan antisense nucleic acid, preferably an antisense oligonucleotide.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an that is an oligoribonucleotide, oligodeoxyribonucleotide,modified oligoribonucleotide, or modified oligodeoxyribonucleotide whichhybridizes under physiological conditions to DNA comprising a particulargene or to an mRNA transcript of that gene and thereby, inhibits thetranscription of that gene and/or the translation of that mRNA.Antisense molecules are designed so as to interfere with transcriptionor translation of a target gene upon hybridization with the target gene.Those skilled in the art will recognize that the exact length of theantisense oligonucleotide and its degree of complementarity with itstarget will depend upon the specific target selected, including thesequence of the target and the particular bases which comprise thatsequence.

It is preferred that the antisense oligonucleotide be constructed andarranged so as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions.

In order to be sufficiently selective and potent for inhibition, suchantisense oligonucleotides should comprise at least 7 (Wagner et al.,Nature Biotechnology 14:840-844, 1996) and more preferably, at least 15consecutive bases which are complementary to the target. Mostpreferably, the antisense oligonucleotides comprise a complementarysequence of 20-30 bases.

Although oligonucleotides may be chosen which are antisense to anyregion of the gene or mRNA transcripts, in preferred embodiments theantisense oligonucleotides correspond to N-terminal or 5′ upstream sitessuch as translation initiation, transcription initiation or promotersites. In addition, 3′-untranslated regions may be targeted. The3′-untranslated regions are known to contain cis acting sequences whichact as binding sites for proteins involved in stabilising mRNAmolecules. These cis acting sites often form hair-loop structures whichfunction to bind said stabilising proteins. A well known example of thisform of stability regulation is shown by histone mRNA's, the abundanceof which is controlled, at least partially, post-transcriptionally.

The term “antisense oligonucleotides” is to be construed as materialsmanufactured either in vitro using conventional oligonucleotidesynthesising methods which are well known in the art or oligonucleotidessynthesised recombinantly using expression vector constructs. Modifiedoligonucleotide is construed in the following manner. The term “modifiedoligonucleotide” as used herein describes an oligonucleotide in which;

-   -   i) at least two of its nucleotides are covalently linked via a        synthetic internucleoside linkage (i.e., a linkage other than a        phosphodiester linkage between the 5′ end of one nucleotide and        the 3′ end of another nucleotide). Alternatively or preferrably        said linkage may be the 5′ end of one nucleotide linked to the        5′ end of another nucleotide or the 3′ end of one nucleotide        with the 3′ end of another nucleotide; and/or    -   ii) a chemical group not normally associated with nucleic acids        has been covalently attached to the oligonucleotide or        oligoribonucleotide. Preferred synthetic internucleoside        linkages are phosphorothioates, alkylphosphonates,        phosphorodithioates, phosphate esters, alkylphosphonothioates,        phosphoramidates, carbamates, phosphate triesters, acetamidates,        peptides, and carboxymethyl esters.        The term “modified oligonucleotide” also encompasses        oligonucleotides with a covalently modified base and/or sugar.        For example, modified oligonucleotides include oligonucleotides        having backbone sugars which are covalently attached to low        molecular weight organic groups other than a hydroxyl group at        the 3′ position and other than a phosphate group at the 5′        position. Thus modified oligonucleotides may include a        2′-O-alkylated ribose group. In addition, modified        oligonucleotides may include sugars such as arabinose instead of        ribose. Modified oligonucleotides also can include base analogs        such as C-5 propyne modified bases (Wagner et al., Nature        Biotechnology 14:840-844, 1996).

The present invention, thus, contemplates pharmaceutical preparationscontaining natural and/or modified antisense molecules that arecomplementary to and, under physiological conditions, hybridizable withnucleic acids encoding proteins the regulation of which results inbeneficial therapeutic effects, together with pharmaceuticallyacceptable carriers (eg polymers, liposomes/cationic lipids).

Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardphysiologically and/or pharmaceutically acceptable carriers which areknown in the art (eg liposomes). The compositions should be sterile andcontain a therapeutically effective amount of the antisenseoligonucleotides for administration to a patient. The term“pharmaceutically acceptable” means a non-toxic material that does notinterfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism.

In yet a still further preferred method of the invention said nucleicacid is a double stranded RNA molecule (RNA). A technique tospecifically ablate gene function is through the introduction of doublestranded RNA, also referred to as inhibitory RNA (RNAi), into a cellwhich results in the destruction of mRNA complementary to the sequenceincluded in the RNAi molecule. The RNAi molecule comprises twocomplementary strands of RNA (a sense strand and an antisense strand)annealed to each other to form a double stranded RNA molecule. The RNAimolecule is typically derived from exonic or coding sequence of the genewhich is to be ablated. In a preferred method of the invention thelength of the RNAi molecule is between 100 bp-1000 bp. More preferablystill the length of RNAi is selected from about 100 bp; 200 bp; 300 bp;400 bp; 500 bp; 600 bp; 700 bp; 800 bp; 900 bp; or 1000 bp. Morepreferably still said RNAi is at least 1000 bp.

In a further preferred method of the invention said RNAi is derived froman exon.

Alternatively said RNAi molecule is derived from intronic sequences orthe 5′ and/or 3′ non-coding sequences which flank coding/exon sequencesof genes. Recent studies suggest that RNAi molecules ranging from100-1000 bp derived from coding sequence are effective inhibitors ofgene expression. Suprisingly, only a few molecules of RNAi are requiredto block gene expression which implies the mechanism is catalytic. Thesite of action appears to be nuclear as little if any RNAi is detectablein the cytoplasm of cells indicating that RNAi exerts its effect duringmRNA synthesis or processing.

In yet a further preferred method of the invention said RNAi moleculescomprise modified ribonucleotide bases. It will be apparent to oneskilled in the art that the inclusion of modified bases, as well as thenaturally occuring bases cytosine, uracil, adenosine and guanosine, mayconfer advantageous properties on RNAi molecules containing saidmodified bases. For example, modified bases may increase the stabilityof the RNAi molecule thereby reducing the amount required to produce adesired effect.

The exact mechanism of RNAi action is unknown although there aretheories to explain this phenomenon. For example, all organisms haveevolved protective mechanisms to limit the effects of exogenous geneexpression. For example, a virus often causes deleterious effects on theorganism it infects. Viral gene expression and/or replication thereforeneeds to be repressed. In addition, the rapid development of genetictransformation and the provision of transgenic plants and animals hasled to the realisation that transgenes are also recognised as foreignnucleic acid and subjected to phenomena variously called quelling(Singer and Selker, Curr Top Microbiol Immunol. 1995;197:165-77), genesilencing (Matzke and Matzke, Novartis Found Symp. 1998;214:168-80;discussion 181-6. Review) and co-suppression (Stam et. al., Plant J.2000;21(1):27-42.

Initial studies using RNAi used the nematode Caenorhabditis elegans.RNAi injected into the worm resulted in the disappearance ofpolypeptides corresponding to the gene sequences comprising the RNAimolecule(Montgomery et. al., 1998; Fire et. al., 1998). More recentlythe phenomenon of RNAi inhibition has been shown in a number ofeukaryotes including, by example and not by way of limitation, plants,trypanosomes (Shi et. al., 2000) Drosophila spp. (Kennerdell andCarthew, 2000). Recent experiments have shown that RNAi may alsofunction in higher eukaryotes. For example, it has been shown that RNAican ablate c-mos in a mouse ooctye and also E-cadherin in a mousepreimplanation embryo (Wianny and Zernicka-Goetz, 2000).

In a yet further preferred method of the invention said nucleic acid isa ribozyme. A ribozyme is a catalytic RNA which is well known in theart. A ribozyme comprises a catalytic core having flanking sequencesadjacent to the sequence which hybridises to the substrate RNA. Thesimplest catalytic core is an RNA motif known as a hammerhead. Since thediscovery of catalytic RNA there has been a desire to design ribozymeswhich have a targetted gene function such that viral mRNA and diseasegene mRNA's can be selectively ablated. For example, U.S. Pat. No.6,069,007 discloses ribozymes active against HIV1 mRNA and their use inAIDS therapy. U.S. Pat. No. 6,087,172 discloses ribozymes designed toablate mRNA encoding IL-15, an interleukin invloved in rheumatoidarthritis. U.S. Pat. No. 6,077,705 discloses a method of gene therapy toinhibit the expression of mutated genes combined with the replacement ofthe mutated gene, in this example α-1-antitrypsin, with a wild-typecopy.

Modes of Administration/Treatment:

The formulations of the present invention are useful for enhancing thetransfer of nucleic acids into tissues. It will be apparent to oneskilled in the art that solutions according to the invention may beintroduced into an animal subject in a variety of ways includingenterally (orally, rectally or sublingually) or parenterally(intravenously, subcutaneously, or by inhalation). The solutions may beprovided to the mammal by implanted catheters, In the preferred practiceof the invention, the solutions are instilled into a body cavity tofacilitate transduction of the surrounding tissues. Examples of suchbody cavities into which the solutions may be provided for the deliveryof nucleic acids include the peritoneal cavity, pleural cavity, and theabdominal cavity. Additionally the solutions may be provided in otherfluid containing spaces such as cerebral spinal fluid, joints, thecolon, the bladder, the eye and gall bladder. It will also be apparentto one skilled in the art that the solutions can be administeredsimultaneously, (as an admixture), separately or sequentially to ananimal.

According to a further aspect of the invention there is provided acomposition comprising dextrin, a sugar, divalent cation and a nucleicacid molecule. Preferably said composition is for use in the delivery ofnucleic acid for gene therapy. In the preferred practice of theinvention, this procedure is employed in conjunction with recombinantadenoviral therapy for the treatment of human cancers. In accordancewith conventional oncology practice, patients are dosed at the maximumtolerated dose of the therapeutic agent. In the course of clinicalinvestigation, a dose of 7.5×10¹³ recombinant adenoviral particles waswell tolerated in human subjects. Clinical experience with replicationdeficient recombinant adenoviruses expressing p53 has indicated that acourse of therapy of injection of approximately 7.5×10¹³ recombinantviral particles for a period of 5 day course of therapy repeated monthlyup to five months is effective in the treatment of ovarian cancer inhuman beings.

In a particularly preferred embodiment of the present invention, aformulation of the present invention comprising a replication deficientrecombinant adenovirus encoding p53 is instilled into the peritoneum forthe treatment of ovarian cancer. A typical course of therapy with thisagent involves administration of 7.5×10¹³ viral particles each day for aperiod of 5 days. A typical clinical protocol for the treatment ofovarian cancer using the A/C/N/53 virus calls involves a typical 5 daycourse of therapy described above in conjunction with the administrationof the chemotherapeutic agents carboplatin and paclitaxel. In thepreferred practice of the invention the mammal is a human being whichreceives three or more courses of therapy, preferably 5-6 courses oftherapy, with intervening rest periods. Modifications to this procedurefor therapeutic viruses other than adenovirus will be readily apparentto the skilled artisan

The formulations of the present invention may further compriseadditional carriers, excipients or diluants. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc. The concentration of the compositions of the invention inthe pharmaceutical formulations can vary widely, i.e., from less thanabout 0.1%, usually at or at least about 2% to as much as 20% to 50% ormore by weight, and will be selected primarily by fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected.

EXAMPLES

The following examples are merely illustrative of the practice of thepresent invention and are not intended to limit the scope thereof.

Example 1 Enhancement of rAd-Mediated Transgene Expression in Rabbits(Intraperitoneal Administration) Using an Icodextrin Formulation

In order to evaluate the ability to enhance transgene expression from arecombinant adenoviral vectors, an experiment was conducted to comparethe relative levels of transgene expression. A recombinant adenoviralvector encoding the beta-galactosidase gene (rAd-bgal) was prepared insubstantial accordance with the teach of Gregory, et al., U.S. Pat. No.5,932,210. The following solutions were preparedin a volume of 100 ml.Solution Components A 1 × 10⁹ particles/ml of rAd-bgal in 15% w/vicodextrin containing 3% sucrose, 2 mM magnesium, 95 mM sodium, 1.75 mMcalcium, 59 mM chloride, 40 mM lactate B 1 × 10⁹ particles/ml ofrAd-bgal, 3% sucrose, 2 mM magnesium chloride in phosphate bufferedsalineTen female New Zealand white rabbits were anaesthetized withketamine/xylazine and the foregoing solutions instilledintraperitoneally. The solution was allowed to incubate for 1 hour (atdorsal side) and one hour (at ventral side). The animals were sacrifiedand biopsies of the peritoneal wall were harvested. Levels of viral RNAin harvested tissues was assayed using RT-PCR. The results of transgenespecific RNA concentrations isolated from the peritoneal wall arepresented in FIG. 1 of the accompanying drawings. As can be seen fromthe data presented addition of 15% icodextrin to the virus buffersolution (solution A) resulted in a marked increase in transgeneexpression relative to the buffer control solution alone (solution B).

Example 2 Efficacy of Icodextrin rAd-p53 Formulation in Murine XenograftProstate Cancer Model

In order to demonstrate that the icodextrin containing formulations ofrecombinant adenoviruses provide an enhanced therapeutic effect, aexperiment was conducted to compare the anti-tumor efficacy ofreplication deficient recombinant adenoviral vectors encoding the p53tumor suppressor gene (“rAd-p53”). The efficacy of the vectors wascompared in a murine xenograft prostate cancer model as described inPaine-Murrieta G D et al. Cancer Chemother Pharmacol 1997, 40: 209.Increased survival was used as the measure of efficacy.

The rAd-p53 vector designated ACN53 was prepared in substantialaccordance with the teaching of Gregory, et al., U.S. Pat. No.5,932,210. PC-3 prostate cancer cells were obtained from the AmericanType Culture Collection, Bethesda Md. under accession number CRL-1435.Fifty-one female nude mice, approximately 5 weeks old were obtained fromHarlan Laboratories. Approximately 5×10⁶ PC3 cells in a volume of 0.2 mlof HBSS-FBS (Hanks Balanced Salt Solution w/10% fetal bovine serum; HBSSwas obtained from Fisher Scientific, FBS was obtained from BioWhittaker)were injected intraperitoneally into each animal. The cells were allowedto establish tumors for a period of nine days prior to the initiation oftreatment.

Seven different formulations were prepared in accordance with Table 2below: TABLE 2 Formulations vPBS (1) vICO (2) Name Description(microliters) (milliliters) virus (3) (microliters) A 15% vICO control 05 0 B 1 × 10¹⁰ PN ACN53; 15% vICO 549 7.2 251 C 1 × 10¹⁰ PN ACN53; vPBS7749 0 251 D 1 × 10⁹ PN ACN53; 15% vICO 775 7.2 25.1 E 1 × 10⁹ PN ACN53;vPBS 7975 0 25.1 F 1 × 10⁸ PN ACN53; 15% vICO 775 7.2 2.51* G 1 × 10⁸ PNACN53; vPBS 7975 0 2.51*Notes:(1) “vPBS” solution is a sterile solution 3% sucrose, 2 mM MgCl₂ inphosphate buffered saline, pH7.4.(2) “vICO” is a sterile 15% icodextrin solution 95 millimolar Na⁺, 1.75millimolar Ca⁺⁺, 2.0 millimolar Mg⁺⁺, 59 millimolar Cl⁻, 40 millimolarlactate, 88 millimolar sucrose, 160 g/liter icodextrin, having a finalosmolarity of 285.75.(3) “virus” refers to a stock virus suspension containing 3.19 × 10¹¹ACN53 viral particles per milliliter. As it is difficult to measure 2.51microliters with conventional equipment, formulations F and G wereprepared by the addition of 25.1 microliters of a 1:10 dilution of thestock viral suspension in vPBS to achieve the equivalent of 2.51microliters of virus suspension.

The animals were divided up into eight treatment groups as more fullydescribed in Table 3 below: TABLE 3 Treatment Groups Total ACN53 Group nFormulation Dose (particles) 1 5 untreated — 2 4 A — 3 7 B 5 × 10¹⁰ 4 7C 5 × 10¹⁰ 5 7 D 5 × 10⁹ 6 7 E 5 × 10⁹ 7 7 F 5 × 10⁸ 8 7 G 5 × 10⁸

Treatment was initiated on Day 9. (Note: All treatment days denoted by“Day” mentioned herein are referenced from the date of injection of thePC-3 cells) All animals appeared healthy upon initiation of thetreatment regimen. Other than the untreated control group 1, each groupwas provided a treatment regimen consisting of treatments eachconsisting of a single intraperitoneal injection of 1.0 ml of theappropriate formulation on Days 9, 11, 14, 15 and 18 following injectionof tumor cells. Upon completion of the treatment regimen, the animalswere randomly caged and monitored daily (blinded) for sick animals ascharactized by visible loss of body weight hunched back, and sedation.Sick animals were sacrificed and examined gross pathologically. Animalnumber, date of sacrifice (or found dead) and gross pathologicalfindings were recorded. The results are summarized in Table 4 below:TABLE 4 Results of PC-3 Tumor Model Survival Time (Days) GroupFormulation median minimum maximum 1 untreated 30 23 33 2 A 30 21 33 3 B53 30 100* 4 C 33 21 42 5 D 36 21 55 6 E 21 21 36 7 F 36 21 42 8 G 36 2139*the experiment was concluded on Day 100Days of survival after injection were plotted using a Kaplan Meiersurvival and the results of treatment groups 1-4 are presentedgraphically in FIG. 2 of the attached drawings. Comparison among groupswas performed using the Logrank Test (Statview Software) Differenceswere considered significant if p<0.05.

As can be seen from the data presented, treatment with ACN53formulations containing icodextrin resulted in a statisticallysignificant prolongation of survival as compared to ACN53 formulated invPBS or controls at a viral dose of 5×10¹⁰ viral particles. One animalreceiving the maximal viral dose in icodextrin was free of clinicalsigns of tumor growth upon completion of the study (Day 100). Thisanimal had minimal tumor growth in the peritoneal cavity indicated thatthe animal was injected with tumor cells and that tumor formed but thattumor growth was inhibited.

Example 3 Efficacy of Icodextrin rAd-p53 Formulation in Murine XenograftOvarian Cancer Model

The effect of icodextrin containing adenoviral formulations containing(Formulations B and C in Table 2 above) was evaluated in a murineintraperitoneal xenograft model of human ovarian cancer. The experimentwas conducted in substantial accordance with the teaching of Example 2above with the following variations. Animals were inoculatedintraperitoneally with 1×10⁷ human MDL-H2774 ovarian cancer cells (ATCCCRL-10303) cells in 0.2 ml of HBSS. The animals were dosedintraperitoneally with 0.5 ml formulations B and C above on Days 2, 5, 8and 12. Control groups were treated as in Example 2. Similarprolongation of survival was observed in this model system withadenovirus formulations comprising icodextrin.

Example 4 vIco Enhanced Efficacy of an Oncolytic Adenovirus in anOrthotopic Model of Human Ovarian Cancer

A murine model of human ovarian cancer was employed to evaluate theeffect of icodextrin containing formulations of a conditionallyreplicating adenoviral vector. The model was established in nude mice(20-25 g commercially available from Harlan, Indianapolis, Ind.) by theadministration of a single intraperitoneal injection of a suspension of1×10⁷ MDA H2774 human ovarian cancer cells (available from the AmericanType Culture Collection under Accession Number CRL-10303) in 0.5 ml inHank's Balanced Salt Solution (HBSS). Tumors were permitted to grow for7 days and the mice were evaluated for the presence of palpable tumors.Those mice evidencing tumors were separated into groups for treatment.

A conditionally replicating recombinant adenoviral vector designatedK9TB was prepared in substantial accordance with the teaching ofRamachandra, et al. (PCT International Publication Number WO 00/22137published Apr. 20, 2000). The K9TB virus is a conditionally replicatingvirus containing a deletion of a amino acids 4-25 of the E1a region, ap53 response element driving expression of the E2F-Rb fusion protein(Antelman, et al., U.S. Pat. No. 6,074,850 issued Jun. 13, 2000)inserted into the E3 region.

The following formulations provided in Table 5 below were prepared forevaluation in the tumor model described above. TABLE 5 Formulations vPBS(1) vICO (2) virus (3) Name Description (microliters) (milliliters)(microliters) H 15% vICO control — 3 — I vPBS control 3 — — J 4.5 × 10⁸PN K9TB; 3.757 — 743 vPBS K 4.5 × 10⁸ PN K9TB; — 3.757 743 15% vICONotes:(1) “vPBS” solution is a sterile solution 3% sucrose, 2 mM MgCl₂ inphosphate buffered saline, pH 7.4.(2) “vICO” is a sterile 15% icodextrin solution 95 millimolar Na⁺, 1.75millimolar Ca⁺⁺, 2.0 millimolar Mg⁺⁺, 59 millimolar Cl⁻, 40 millimolarlactate, 88 millimolar sucrose, 160 g/liter icodextrin.(3) “virus” refers to a 1:1000 dilution of a stock virus suspensioncontaining 6.06 × 10¹¹ K9TB viral particles per milliliter.

The tumor bearing animals were segregated into the following groups fortreatment: TABLE 6 Treatment Groups Total K9TB Dose Group n Formulation(particles) 1 4 H — 2 4 I — 3 7 J 2 × 10⁸ 4 7 K 2 × 10⁸

Each group was provided a treatment regimen consisting of a singleintraperitoneal injection of 0.5 ml of the appropriate formulation onDays days 7, 9, 12, and 14 following injection of tumor cells. Uponcompletion of the treatment regimen, the animals were randomly caged andmonitored daily (blinded) for moribund animals as characterized byvisible loss of body weight hunched back, and sedation. Moribund animalswere sacrificed and examined gross pathologically. Animal number, dateof sacrifice (or found dead) and gross pathological findings wererecorded and the results summarized in Table 7 below. TABLE 7 Results ofH2774 Ovarian Tumor Model Survival Time (Days) Group Formulation meanminimum maximum 1 H 28.5 28 29 2 I 28.3 28 29 3 J 41.0 36 47 4 K 55.7 41 80**the experiment was concluded on Day 80Days of survival after injection were summarized using a Kaplan-Meierplot and the results of treatment groups 1-4 are presented graphicallyin FIG. 3 of the attached drawings. Comparison among groups wasperformed using the Logrank Test (Statview Software). Differences wereconsidered significant if p<0.05.

A graphical representation of the data is presented in FIG. 3 of theattached drawings. Formulation H/Treatment Group 2 is represented by X,Formulation I/Treatment Group 1 is represented by squares, FormulationJ/Treatment Group 3 is represented by circles; and FormulationL/Treatment Group 4 is represented by triangles.

As can be seen from the data presented, the treatment of tumor bearingmice with the recombinant adenoviral vector K9TB in both the vIco andvPBS formulations prolonged survival compared to the vehicle controls.However, K9TB formulated in vIco demonstrated a prolongation of survivalcompared to K9TB formulated in vPBS (p<0.01, n=7 animals/treatmentgroup).

1. A method to deliver nucleic acid to a cell wherein the nucleic acidis carried in a solution comprising dextrin and at least one sugar, theosmolarity of which corresponds substantially to the physiologicalosmolarity of the milieu surrounding the cell.
 2. The method accordingto claim 1 wherein the molecular weight of the dextrin is in the rangefrom about 1,000-200,000.
 3. The method according to claim 2 wherein themolecular weight of the dextrin is between about 2,000-55,000.
 4. Themethod according to claim 3 wherein the dextrin contains more than about15% w/v of polymers of a degree of polymerisation greater than
 12. 5.The method according to claim 4 wherein the dextrin contains more thanabout 50% w/v of polymers of a degree of polymerisation greater than 12.6. The method according to claim 5 wherein the dextrin is present in thesolution in an amount of less than about 20% w/v.
 7. The methodaccording to claim 6 wherein the dextrin is present in the solution inan amount selected from about: 1% w/v; 2% w/v; 3% w/v; 4% w/v; 5% w/v;6% w/v; 7% w/v; 8% w/v; 9% w/v; 10% w/v; 11% w/v; 12% w/v; 13% w/v; 14%w/v; 15% w/v; 16% w/v; 17% w/v; 18% w/v;. 19% w/v; 20% w/v.
 8. Themethod according to claim 7 wherein the dextrin is present from 1-5%w/v.
 9. The method according to claim 8 wherein the dextrin ispreferably about 4% w/v.
 10. The method according to any of claim 1wherein the sugar is a disaccharide.
 11. The method according to claim10 wherein the amount of dissacharide is between about 1 and 10% w/v.12. The method according to claim 11 wherein the dissacharide is anamount of between about 2 and 5% w/v.
 13. The method according to claim12 wherein the dissacharide is sucrose and the amount of sucrose isabout 3% w/v.
 14. The method according to claim 1 wherein the amount ofdextrin is about between 2%-20% w/v and the amount of sucrose is betweenabout 1-10% w/v.
 15. The method according to claim 14 wherein the amountof dextrin is about 15% w/v and the amount of sucrose is about 3% w/v.16. The method according to claim 15 wherein the amount of dextrin isabout 4% w/v and the amount of sucrose is about 3% w/v.
 17. The methodaccording to claim 1 wherein the solution further comprises a divalentcation.
 18. The method according to claim 17 wherein the divalent cationis in a concentration of at least 0.2 mM.
 19. The method according toclaim 19 wherein the divalent cation concentration is between 0.2-3.0mM.
 20. The method according to claim 19 wherein the divalent cation isprovided by MgCl₂ and the concentration is about 2.0 mM.
 21. The methodaccording to claim 20 wherein the solution comprises about 4% w/vdextrin, about 3% w/v sucrose and about 2.0 mM MgCl₂.
 22. The methodaccording to claim 1 wherein the nucleic acid molecule is a vector. 23.The method according to claim 22 wherein the vector is adapted foreukaryotic expression.
 24. The method according to claim 22 wherein thevector is a recombinant virus derived from adenovirus, retrovirus,adeno-associated virus, herpesvirus, lentivirus, vaccinia virus, orbaculovirus.
 25. The method of claim 24 wherein said vector is anadenovirus.
 26. The method of claim 25 wherein said adenovirus furtherencodes an exogenous transgene.
 27. The method of claim 26 wherein saidtransgene is a tumor suppressor gene.
 28. The method of claim 27 whereinsaid tumor suppressor gene is p53.
 29. The method of claim claims 24 to26 wherein said vector is replication competent.
 30. The method of claim29 wherein said vector is a conditionally replicating replicationcompetent vector.
 31. A composition for delivery of a recombinant viralvector to a cell comprising a recombinant viral vector in a solutioncomprising dextrin and at least one sugar, the osmolarity of whichcorresponds substantially to the physiological osmolarity of the milieusurrounding the cell.
 32. The composition of claim 31 wherein therecombinant viral vector is derived from adenovirus, retrovirus,adeno-associated virus, herpesvirus, lentivirus, vaccinia virus, orbaculovirus.
 33. The composition of claim 32 wherein the viral vector isderived from adenovirus.
 34. The composition of claim 33 wherein theviral vector is a selectively-replicating replication competentadenoviral vector.
 35. A pharmaceutical formulation comprising arecombinant adenoviral vector, a dextrin, a sugar and a divalent cation.36. A method to deliver nucleic acid to a cell comprising: providing asolution of dextrin, a sugar, a divalent cation and nucleic acid andcontacting the solution with a cell to be transfected or transformed.